Immune checkpoint blockade and its combination therapy with small-molecule inhibitors for cancer treatment

Initially understood for its physiological maintenance of self-tolerance, the immune checkpoint molecule has recently been recognized as a promising anti-cancer target. There has been considerable interest in the biology and the action mechanism of the immune checkpoint therapy, and their incorporation with other therapeutic regimens. Recently the small-molecule inhibitor (SMI) has been identified as an attractive combination partner for immune checkpoint inhibitors (ICIs) and is becoming a novel direction for the field of combination drug design. In this review, we provide a systematic discussion of the biology and function of major immune checkpoint molecules, and their interactions with corresponding targeting agents. With both preclinical studies and clinical trials, we especially highlight the ICI + SMI combination, with its recent advances as well as its application challenges.     

The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.     

Slx4 and Rtt107 control checkpoint signalling and DNA resection at double-strand breaks

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5′ strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.     

Integrin–ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium

Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. We recently identified Diedel, a secreted protein/cytokine, as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. Here, we show that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, we uncovered a resilience mechanism of epithelial tissues, mediated by Integrin–ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, we found that resilient epithelial cells, enriched for Diedel–Integrin–ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin–ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin–ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.

A key feature of the resilience mechanisms that underlie the control of epithelial tissue damage is the balance between cell death and survival. This study shows that the anti-oxidant enzyme catalase can relocate to membranes in order to promote the resilience of the Drosophila midgut epithelium, synergizing with integrin-ECM interactions to prevent the spread of cell death.     

Localization of the Drosophila Rad9 protein to the nuclear membrane is regulated by the C-terminal region and is affected in the meiotic checkpoint

Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. It was shown previously that the C-terminal end of the human Rad9 protein, which contains a nuclear localization sequence (NLS) nearby, is critical for the nuclear transport of Rad1 and Hus1. In this study, we show that in Drosophila, Hus1 is found in the cytoplasm, Rad1 is found throughout the entire cell and that Rad9 (DmRad9) is a nuclear protein. More specifically, DmRad9 exists in two alternatively spliced forms, DmRad9A and DmRad9B, where DmRad9B is localized at the cell nucleus, and DmRad9A is found on the nuclear membrane both in Drosophila tissues and also when expressed in mammalian cells. Whereas both alternatively spliced forms of DmRad9 contain a common NLS near the C terminus, the 32 C-terminal residues of DmRad9A, specific to this alternative splice form, are required for targeting the protein to the nuclear membrane. We further show that activation of a meiotic checkpoint by a DNA repair gene defect but not defects in the anchoring of meiotic chromosomes to the oocyte nuclear envelope upon ectopic expression of non-phosphorylatable Barrier to Autointegration Factor (BAF) dramatically affects DmRad9A localization. Thus, by studying the localization pattern of DmRad9, our study reveals that the DmRad9A C-terminal region targets the protein to the nuclear membrane, where it might play a role in response to the activation of the meiotic checkpoint.     

Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation

Assessment of cell membrane integrity is one of the most widely used methods to measure post-cryopreservation viability of cells such as human umbilical vein endothelial cells (HUVECs). However, an evaluation of cell function provides a better measure of cell quality following cryopreservation. The tube formation assay mimics angiogenesis in vitro and can be used to quantitate the ability of endothelial cells to form capillary-like tubular structures when cultured on reconstituted basement membrane (Matrigel). We compared the membrane integrity (measured by flow cytometry) and tube forming ability of HUVEC suspensions exposed to 10% dimethyl sulfoxide (Me₂SO), cooled at 1 °C/min to various sub-zero temperatures, plunged directly into liquid nitrogen, stored for an hour, and thawed rapidly. We found that as membrane integrity increased so did the various parameters associated with the extent of in vitro angiogenesis; however, in comparison to fresh cells with a similar percentage of membrane-intact cells, the extent of tube formation, expressed as total tube length, is significantly lower in previously frozen cells for the lower range of post-thaw membrane integrities. Our findings underscore the value of an assay that quantifies a specific function that a cell is known to perform in vivo to measure the success of cryopreservation protocols.     

Induction of apoptosis by cooperative bacteria in the morphogenesis of host epithelial tissues

 Associations with pathogenic bacteria have recently been shown to initiate apoptotic programs in the cells of their animal hosts, where host cell death is hypothesized to be a response of the immune system, either initiated as a mechanism of host defense or bacterial offense. In this study, we present evidence that bacterial initiation of apoptosis is neither restricted to pathogenesis nor to the initation of an immune response. In the cooperative association between the sepiolid squid Euprymna scolopes and the luminous bacterium Vibrio fischeri, the bacteria induce a dramatic morphogenesis of the host tissues during the first few days of interaction between these partners. The most striking change is the bacteria-triggered loss of an extensive superficial epithelium that potentiates the infection process. Our analyses of these tissues revealed that the bacteria induce apoptosis in the cells that comprise this epithelium within hours of the interaction with bacteria. Ultrastructural analysis revealed that after 24 h the integrity of the epithelium had been lost, i.e., the basement membrane had degenerated and the majority of the cells exhibited signs of apoptosis, most notably chromatin condensation. Analysis of these tissues with probes that reveal intracellular acidification showed that the cells first undergo an initial acidification beginning about 6–8 h after exposure to V. fischeri. As determined by end-labeling of DNA fragments, extensive endonuclease activity was detected at approximately 16–20 h post-infection. These data provide evidence that cooperative bacteria can participate in the remodeling of host tissues through the induction of host apoptotic programs. Received: 10 November 1997 / Accepted: 22 April 1998     

Protein kinase C controls activation of the DNA integrity checkpoint

The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.     

Compound genetic ablation of nidogen 1 and 2 causes basement membrane defects and perinatal lethality in mice

Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.     

Genetic interaction between Caenorhabditis elegans teneurin ten-1 and prolyl 4-hydroxylase phy-1 and their function in collagen IV-mediated basement membrane integrity during late elongation of the embryo

Teneurins are a family of phylogenetically conserved proteins implicated in pattern formation and morphogenesis. The sole orthologue in Caenorhabditis elegans, ten-1, is important for hypodermal cell migration, neuronal migration, path finding and fasciculation, gonad development, and basement membrane integrity of some tissues. However, the mechanisms of TEN-1 action remain to be elucidated. Using a genome-wide RNA interference approach, we identified phy-1 as a novel interaction partner of ten-1. phy-1 codes for the catalytic domain of collagen prolyl 4-hydroxylase. Loss of phy-1 significantly enhanced the embryonic lethality of ten-1 null mutants. Double-mutant embryos arrested during late elongation with epidermal defects, disruption of basement membranes, and detachment of body wall muscles. We found that deletion of phy-1 caused aggregation of collagen IV in body wall muscles in elongated embryos and triggered the loss of tissue integrity in ten-1 mutants. In addition, phy-1 and ten-1 each genetically interact with genes encoding collagen IV. These findings support a functional mechanism in which loss of ten-1, together with a reduction of assembled and secreted basement membrane collagen IV protein, leads to detachment of the epidermis from muscle cells during late elongation of the embryo when mechanical stress is generated by muscle contractions.     

Regional differences in the expression of laminin isoforms during mouse neural tube development

Many significant human birth defects originate around the time of neural tube closure or early during post-closure nervous system development. For example, failure of the neural tube to close generates anencephaly and spina bifida, faulty cell cycle progression is implicated in primary microcephaly, while defective migration of neuroblasts can lead to neuronal migration disorders such as lissencephaly. At the stage of neural tube closure, basement membranes are becoming organised around the neuroepithelium, and beneath the adjacent non-neural surface ectoderm. While there is circumstantial evidence to implicate basement membrane dynamics in neural tube and surface ectodermal development, we have an incomplete understanding of the molecular composition of basement membranes at this stage. In the present study, we examined the developing basement membranes of the mouse embryo at mid-gestation (embryonic day 9.5), with particular reference to laminin composition. We performed in situ hybridization to detect the mRNAs of all eleven individual laminin chains, and immunohistochemistry to identify which laminin chains are present in the basement membranes. From this information, we inferred the likely laminin variants and their tissues of origin: that is, whether a given basement membrane laminin is contributed by epithelium, mesenchyme, or both. Our findings reveal major differences in basement composition along the body axis, with the rostral neural tube (at mandibular arch and heart levels) exhibiting many distinct laminin variants, while the lumbar level where the neural tube is just closing shows a much simpler laminin profile. Moreover, there appears to be a marked difference in the extent to which the mesenchyme contributes laminin variants to the basement membrane, with potential contribution of several laminins rostrally, but no contribution caudally. This information paves the way towards a mechanistic analysis of basement membrane laminin function during early neural tube development in mammals.     

Bub1 regulates chromosome segregation in a kinetochore-independent manner

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.     

Ubiquitin in the immune system

Ubiquitination is a post-translational modification process that has been implicated in the regulation of innate and adaptive immune responses. There is increasing evidence that both ubiquitination and its reversal, deubiquitination, play crucial roles not only during the development of the immune system but also in the orchestration of an immune response by ensuring the proper functioning of the different cell types that constitute the immune system. Here, we provide an overview of the latest discoveries in this field and discuss how they impact our understanding of the ubiquitin system in host defence mechanisms as well as self-tolerance.     

Cell cycle re-entry mechanisms after DNA damage checkpoints: Giving it some gas to shut off the breaks!

In order to maintain genetic integrity, cells are equipped with cell cycle checkpoints that detect DNA damage, orchestrate repair, and if necessary, eliminate severely damaged cells by inducing apoptotic cell death. The mitotic machinery is now emerging as an important determinant of the cellular responses to DNA damage where it functions as both the downstream target and the upstream regulator of the G2/M checkpoint. Cell cycle kinases and the DNA damage checkpoint kinases appear to reciprocally control each other. Specifically, cell cycle kinases control the inactivation of DNA damage checkpoint signaling. Termination of a DNA damage response by mitotic kinases appears to be an evolutionary conserved mechanism that allows resumption of cell cycle progression. Here we review recent reports in which molecular mechanisms underlying checkpoint silencing at the G2/M transition are elucidated.     

Collagen XV Inhibits Epithelial to Mesenchymal Transition in Pancreatic Adenocarcinoma Cells

Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth in vitro and to reduce tumor burden and increase survival in vivo support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated in vitro by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone.     

Keeping Tumors in Check: A Mechanistic Review of Clinical Response and Resistance to Immune Checkpoint Blockade in Cancer

Immune checkpoints are a diverse set of inhibitory signals to the immune system that play a functional role in adaptive immune response and self-tolerance. Dysregulation of these pathways is a vital mechanism in the avoidance of immune destruction by tumor cells. Immune checkpoint blockade (ICB) refers to targeted strategies to disrupt the tumor co-opted immune suppression to enhance anti-tumor immunity. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) are two immune checkpoints that have the widest range of antibody-based therapies. These therapies have gone from promising approaches to Food and Drug Administration-approved first- and second-line agents for a number of immunogenic cancers. The burgeoning investigations of ICB efficacy in blood and solid cancers have underscored the importance of identifying the predictors of response and resistance to ICB. Identification of response correlates is made complicated by the observations of mixed reactions, or different responses in multiple lesions from the same patient, and delayed responses that can occur over a year after the induction therapy. Factors that can influence response and resistance in ICB can illuminate underlying molecular mechanisms of immune activation and suppression. These same response predictors can guide the identification of patients who would benefit from ICB, reduce off-target immune-relate adverse events, and facilitate the use of combinatorial therapies to increase efficacy. Here we review the underlying principles of immune checkpoint therapy and results of single-agent ICB clinical trials, and summarize the predictors of response and resistance.     

Molecular dissection of ARP1 regions required for nuclear migration and cell wall integrity checkpoint functions in Saccharomyces cerevisiae

The dynactin complex is one of the components required for the regulation of the cell wall integrity checkpoint, which ensures the completion of cell wall remodeling before mitosis. The core of the dynactin complex is a backbone filament composed of monomers of an actin-related protein, Arp1, which is also involved in nuclear migration. To examine the molecular basis for the dual functions of the dynactin core subunit Arp1p in yeast, we constructed 32 mutated arp1 alleles. We assessed the effects of the mutations on cell wall integrity checkpoint and nuclear migration functions and identified four categories of mutants: 1) those showing no change from the wild type; 2) those resulting in a defective cell wall integrity checkpoint but normal nuclear migration; 3) those with a normal cell wall integrity checkpoint but defective nuclear migration; and 4) those defective in both the cell wall integrity checkpoint and nuclear migration functions. Our results show a separation of the two functions in the molecular structure of Arp1p and indicate that a local surface region of Arp1p is important in maintaining the cell wall integrity checkpoint function.     

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.